We have undertaken the following projects: Characterization of O-glycosylation in collagen Aims: 1) Type 1 collagen is the major organic component in bone providing a structural template for mineralization. During collagen biosynthesis, specific hydroxylysine (Hyl) residues become glycosylated in the form of galactosylhydroxylysine (G-Hyl) and glucosyl galactosylhydroxylysine (GG-Hyl). Furthermore, key glycosylated Hyl residues are involved in covalent intermolecular collagen cross-linking. While cross-linking is crucial for fibril formation, the biological relevance of glycosylation and its relationship to cross-linking are not well understood. The extensive analytical characterization of cross-links and their glycosylation is an essential step in elucidating the function of these modifications. In this study, we isolated several major cross-linked peptides from a trypic digest of NaB3H4-reduced bovine bone type I collagen and characterized glycosylation of cross-links structurally and semi-quantitatively by employing nanoscale liquid chromatography - high resolution tandem mass spectrometry (nanoLC/MS/MS) The results show that collagen cross-links in bone are differentially glycosylated, depending on their molecular location (1/2-87 vs. 1-930, 2-933), type- and maturational stage. The data, together with our recent report (Sricholpech et al J Biol Chem 2012), indicates that the extent and pattern of glycosylation may control cross-link maturation. Thus thorough molecular characterization provides critical insights into the function of this glycosylation in bone physiology. 2) In collision induced dissociation (CID), collagen glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides, i.e. in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed. The G- or GG- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. We present here the experimental results obtained in the area of collagen glycopeptide fragmentation. Furthermore, we employ these experimental observations as the basis for quantum mechanics calculations, in order to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides. Characterization of intermediates in DNA repair. Rev1-DNA Crosslinking and Mutant Beta polymerase-DNA Crosslinking. A methodology for the specific enrichment and MS analysis of protein-DNA crosslinks is currently being developed. Additionally, products following crosslinking have been purified by HPLC and by SDS-PAGE. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and electrospray mass spectrometry. Oxidative Stress DMPO. Radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In this work, we have investigated the sites of radicals trapped by DMPO on deoxyribonucleosides and proteins using LC/MS/MS. For proteins: DMPO adducts have been determined for acetylcholinesterase and protein radicals are implicated in heme oxygenase-1 and myeloperoxidase using different protocols to generate radicals. For DNA: DMPO adducts of DNA using various systems is under investigation. DMPO adducts of adenine, guanidine, and cytosine have been confirmed.